The prevalence of clarithromycin-resistant Helicobacter pylori isolates: a systematic review and meta-analysis

Background Knowledge of global clarithromycin (CLA)-resistant rates of Helicobacter pylori (H. pylori) is crucial for decision of the most appropriate eradication therapies with good clinical outcomes. Therefore, this review and meta-analysis aimed to evaluate the global prevalence of the CLA resistance in H. pylori to provide some guidance for selecting the first-line antibiotics. Method A comprehensive search was performed for relevant literature until April 2021 in PubMed, Embase, and Web of Science databases. Freeman-Tukey double arcsine transformation was performed to estimate the weighted pooled prevalence of resistance. Results The meta-analysis included 248 articles. The prevalence of CLA-resistant H. pylori was 27.53% (95% CI [25.41–29.69]). The heterogeneity between reports was significant (I2 = 97.80%, P < 0.01). The resistance rate increased from 24.28% in 2010–2017 to 32.14% in 2018–2021 (P < 0.01). Iran, with 38 articles, has the most report. Nevertheless, Switzerland, Portugal, and Israel had the highest resistance rates (67.16%, 48.11%, and 46.12%, respectively). The heterogeneity between the continents and the antimicrobial susceptibility methods also interpreted standard guidelines and breakpoints was insignificant (P > 0.05). Conclusion Overall CLA resistance rate was 27.53%, worldwide. The difference in CLA resistance rate among the included studies can be due to several reasons such as differences in antibiotic prescription rates in various geographic areas, use of different breakpoints or inaccurate criteria in performed studies, and the emergence of multidrug-resistant (MDR) strains.


INTRODUCTION
Helicobacter pylori is one of the most successful human pathogens that affects approximately 50% of the population worldwide. In developing countries 70% to 90% of the population are infected by this bacterium (Arenas et al., 2019;Kocsmár et al., 2021). H. pylori infection is related to many gastric diseases, such as peptic ulcers, chronic gastritis, uninvestigated and functional dyspepsia and mucosa-associated lymphoid tissue lymphoma, and even increases the risk of gastric cancer (Savoldi et al., 2018). As for the high prevalence of the bacterium and its related diseases, proper treatment is very important. Today, standard treatment is a three-stage drug that consists of an acid neutralizer and two antibiotics, clarithromycin (CLA), and amoxicillin or metronidazole for 14 days (Hosseini et al., 2021).
However, treatment is difficult because the bacterium quickly develops resistance to the few antibiotics known to be effective (Park et al., 2016). The World Health Organization (WHO) has classified it among the 12 most resistant bacteria in the world (Essaidi et al., 2022). The increasing failure rate of eradication treatment due to the appearance of resistant H. pylori strains contributes to the worldwide prevalence of this infection and subsequent inflammatory and neoplastic disorders. Unfortunately, nowadays, the success of this treatment is less than 80% worldwide (Kocsmár et al., 2021;Hussein, Al-Ouqaili & Majeed, 2022).
CLA has been emerged as the basis for H. pylori treatment in combined therapy because of small effect on gastric acidity, its low minimal inhibitory concentration, and relatively good mucosal diffusion (Marques et al., 2020;Nishizawa & Suzuki, 2014). Due to extensive usage of CLA in some geographical regions, global prevalence rate of CLA resistance is increasing (Zou et al., 2020). In developing countries, CLA resistance and frequency of re-infection are factors that contribute to high worldwide prevalence of H. pylori infection and subsequent inflammatory and neoplastic disorders (Alarcón-Millán et al., 2016). In most European countries, as well as the rest of the world, the prevalence of CLA resistance has reached 20%. With rare exceptions, it is no longer recommended to include CLA in empirical treatment in regions where primary resistance to this antibiotic is 20% (Alarcón-Millán et al., 2016;.
Knowledge of global CLA-resistant rates of H. pylori is crucial for decision of the most appropriate eradication therapies with good clinical outcomes. Therefore, the aim of current review and meta-analysis is to evaluation of the global prevalence of the CLA resistance in H. pylori.

Statistical analysis
For the present study, the sample size of isolates for antimicrobial susceptibility testing (AST) and the number of resistances to each antibiotic were used to calculate a weighted pooled resistance and their 95% confidence intervals. In order to prevent the exclusion of studies from the meta-analysis due to 0 or 100 resistance prevalence, the Inverse of Freeman-Tukey double arcsine transformation was conducted using Metaprop command in STATA software (version 17.1). A random-effects model was implemented to estimate pool proportions (Egger et al., 1997;Harbord et al., 2010). The I 2 with a P ≤ 0.05 was used to identify significant heterogeneity. The presence of a small-study effect or publication bias was assessed using Egger's linear regression test and Begg's test (Harbord, Harris & Sterne, 2009). Subgroup analyses were conducted to determine the impact of the country, continent, publication year (2010)(2011)(2012)(2013)(2014)(2015)(2016)(2017)(2018)(2019)(2020)(2021), (AST) (Disc diffusion, Gradient methods), and breakpoints for interpretation of AST results on the variation.

Descriptive statistics
In this research, 19,169 records were acquired in EndNote version 20, a reference manager software. A total of 8,689 duplicated articles were then removed, leaving a total of 247 eligible studies that were included in the systematic review and meta-analysis. The screening and selection presage were summarized in the PRISMA flow chart (Fig. 1). Overly 20,936 H. pylori isolates have been investigated in included articles. More than half of the isolates were investigated in Asia (55.10% Isolated). Although most pieces were from Iran (38 articles), the highest number of isolates among the countries was that investigated from China (32,130 Isolates, 36.52% of total isolates). Description data are summarized in Table 2.

Publication bias
The publication bias was significant by the regression-based Egger test for small-study effects (P = 0.04), but Begg's test for small-study effects was insignificant (P = 0.09).
The Nonparametric trim-and-fill analysis of publication bias also did not change the effect size. The funnel plot also did not have significant evidence of publication bias ( Fig. 2A).

DISCUSSION
Over the past years, the treatment of H. pylori infections has been performed using the standard triple therapy regimen, including CLA, a proton pump inhibitor, with either metronidazole or amoxicillin . However, in recent years, it is revealed that some H. pylori isolates have developed resistance to CLA (Sanches et al., 2016). Therefore, the efficacy of the standard triple therapy regimen is in decline. In 2017, WHO listed the CLA-resistant H. pylori among antibiotic-resistant priority pathogens that need research and development of new antibiotics (Khani, Abadi & Mobarez, 2019). Globally, surveillance and being aware of the frequency of resistance to antibiotics among pathogens is critical, and obtained results can be helpful in different sections such as the design of screening or follow-up programs, and the development of antimicrobial stewardship programs (Azimi et al., 2019;Pormohammad, Nasiri & Azimi, 2019).
In the present systematic review and meta-analysis study, we surveyed and analyzed the worldwide prevalence of CLA resistance among H. pylori isolates from 2010 to 2021. The awareness of CLA resistance among different countries of the world and effective treatment of H. pylori infections are the main goal of the current study. The present systematic review and meta-analysis study included 247 eligible studies from 54 different  countries. Our analyses revealed that the overall prevalence of clarithromycin-resistance H. pylori was 27.53%, worldwide. Resistance to CLA among H. pylori is occur in two different levels including (1) a high level of resistance (MIC more than 64 mg l −1 ) and (2) a low level of resistance (0.5 ≤ MIC ≤ 1 mg l −1 ) (He et al., 2021). Point mutations, multidrug efflux pump systems, and synergistic effect of mutations in genes rpl22 (ribosomal protein L22) and infB (translation initiation factor IF-2) with 23S rRNA point mutations are the main CLA resistance mechanisms among H. pylori isolates (Marques et al., 2020;Li et al., 2021). Moreover, it is presumed that some outer-membrane proteins have a role in CLA resistance in H. pylori isolates (Marques et al., 2020). In the Western world and among developed countries, more than 90% of CLA resistance is related to point mutations in the peptidyl transferase region of the V domain of 23S rRNA gene (Mégraud, 2004). The main point mutations related to CLA resistance are A2142G, A2143G (adenine-to-guanine transition at either position 2142 or 2143), A2142C (adenine-to-cytosine transversion at position 2142), A2115G, A2144T, G2141A, G2144T, T2289C, T2717C, and C2694A Marques et al., 2020;Li et al., 2021). Moreover, hp1181 and hp1184 mutations are associated with CLA resistance (Li et al., 2021). Mutation in the 2142 and 2143 positions leads to restricted resistance and different levels of resistance, respectively .
In the present research, more than half of the included studies were performed in Asia. These results demonstrated that CLA resistance is a main public health issue in most Asian countries. Among studies surveyed CLA resistance rates in 54 different countries, Switzerland (67.16%) and Senegal (0.93%) had the highest and lowest resistance rates, respectively. The high level of CLA resistance can be due to the following reasons: (1) inappropriate prescription and unregulated or widespread use of CLA, and (2) the use of CLA in other infections such as respiratory tract infections or intestinal parasites infections (Chen et al., 2017). Time trend analyses revealed that the CLA-resistant rates among H. pylori isolate increased from 24.28% in 2010-2017 to 32.14% in the 2018-2021 years' period. An increase in CLA resistance rates is an alarming finding. In areas where CLA-resistance is more than 15%, it is recommended to perform susceptibility testing before prescribing the standard triple therapy regimen (Sanches et al., 2016;Abadi, 2017). Combination therapy with other drugs such as tinidazole can be helpful in the treatment of H. pylori infections. It is revealed that CLA combined with tinidazole can reduce the CLA resistance rate, decrease inflammatory reactions, and can effectively eliminate H. pylori infections (He et al., 2021). One of the limitations of this study was that we evaluated the CLA resistance rate only and the other antibiotics were not considered. CONCLUSION Our analysis revealed that CLA resistance rates varied among studies performed in different 54 countries. Altogether, results showed that the overall CLA resistance rate is 27.53%, worldwide. The difference in CLA resistance rate among the included studies can be due to several reasons such as differences in antibiotic prescription rates in various geographic areas, use of different MIC breakpoints or inaccurate criteria in performed studies, and the emergence of multidrug-resistant (MDR) strains. We performed a time trend analysis and the results revealed that the clarithromycin-resistance rates in increasing in recent years. Based on our findings, systematic surveillance, and proper monitoring of CLA resistance rates, as well as monitoring the use of CLA in patients, and performing the CLA susceptibility test before prescription may be critical actions for the inhibition and control of H. pylori infections.

ADDITIONAL INFORMATION AND DECLARATIONS Funding
The authors received no funding for this work.

Competing Interests
Morteza Saki is a PeerJ Academic Editor. The authors declare that they have no competing interests.

Data Availability
The following information was supplied regarding data availability: The raw measurements are available in the Supplemental File.

Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.15121#supplemental-information.